Highly sensitive bioluminescence and fluorescence imaging system for large fields of view

主要设备将用作成像系统，在分子植物研究中在整个器官和生物体水平上读取转基因荧光或生物发光报告线。所申请的设置将使成像在一个新的规模，这将允许监测系统的反应，例如生物或非生物应力的活样品在一个有机的背景。研究的过程是相关的了解植物的生理反应，非生物和生物胁迫条件下，因为它们越来越多地发生与渐进的气候变化。所用的转基因报道细胞系表达例如靶向不同亚细胞区室的遗传编码的生物传感器（响应生理参数而特异性改变其性质的荧光蛋白），或与荧光素酶作为报道细胞的启动子或蛋白质融合物。这种实验方法允许监测亚细胞过程，如氧化还原状态的局部变化或在系统背景下基因表达的调节。利用本提案中申请的主要设备，可以在大视场（约100米）内记录整个生物体（如植物）的生物发光或荧光。20厘米）。为此，将使用冷却至-90 ° C的高性能CCD相机（16位），该相机具有必要的灵敏度和非常低的背景噪声（暗电流）。相机设置位于一个遮光盒中，该盒还包含荧光所需的照明。为了覆盖尽可能多的荧光蛋白和基因编码的生物传感器，必须包括合适的激发和发射滤光片。仪器和采集参数的调整通过外部PC上的一个专用软件进行控制。拟议的设备将首次在德国的植物科学中使用。

The major equipment will be used as an imaging system to read out transgenic fluorescent or bioluminescent reporter lines at the level of whole organs and organisms in molecular plant research. The applied for setup will enable imaging on a new scale, which will allow for the monitoring of systemic reactions to e.g. biotic or abiotic stress on the living sample in an organismic context. The processes studied are relevant for understanding the physiological responses of plants to abiotic and biotic stress conditions, as they increasingly occur with progressive climate change. The transgenic reporter lines used express e.g. genetically encoded biosensors (fluorescent proteins that change their properties specifically in response to a physiological parameter) with targeting to different subcellular compartments, or promoter or protein fusions with luciferase as reporter. This experimental approach allows monitoring of subcellular processes such as local changes in redox status or regulation of gene expression in a systemic context. With the major equipment applied for in this proposal, the bioluminescence or fluorescence of whole organisms such as plants can be recorded in large fields of view (approx. 20 cm). For this purpose, a high-performance CCD camera (16 bit) cooled to -90°C will be used, which has the necessary sensitivity and very low background noise (dark current). The camera setup is located in a light-proof box, which also contains the necessary illumination for fluorescence. To cover as many fluorescent proteins and genetically encoded biosensors as possible, suitable excitation and emission filters must be included. The adjustment of the instrument and acquisition parameters are controlled via a manufacturer-specific software from an external PC. The proposed device would be used for the first time in Germany in plant sciences.